Derivatives of porphyrins, their process of preparation and their use for treating viral infections

ABSTRACT

The present invention concerns metallated porphyrin derivatives as ligands of G-quadruplex and their novel use as anti-viral agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.15/772,355, having a filing date of Apr. 30, 2018, which was a 371application of International application PCT/EP2016/076127 filed on Oct.28, 2016, which claimed the benefit of European patent application EP15306737.6, filed Oct. 30, 2015, all of said applications incorporatedherein by reference.

The present invention concerns derivatives of porphyrins, theirpreparation and their application in therapeutics.

More specifically, this application is related to the use of porphyrinderivatives as G-quadruplex ligands to inhibit viral infections, such asHIV, more particularly to inhibit HIV-1 replication cycle.

Guanine rich RNA or DNA sequences are capable of folding and adoptingfour stranded structures called G-quadruplexes, or “G4” These unusualnucleic acid structures are based on the stacking of 2, 3 or 4 tetrads;each of which is composed of four guanines connected by 8 hydrogenbonds. These tetrads are stabilized by the presence of a central cation,K⁺ or Na⁺, abundantly present in the cellular environment, and which iscoordinated with the oxygens of the carbonyl groups. Numerousthermodynamic studies have shown that these structures are very stable.They often have thermal denaturation temperatures above 50° C., and someare stable at 90° C. Once formed, some G4 such as the one formed by thec-myc promoter sequence, have a very long half-life and can withstandthe annealing in the presence of high excess of their complementarystrand (up to 50 times). Polymorphism, robustness and fast folding aresome of the intrinsic characteristics of the G4 which strongly suggest abiological role.

Several bioinformatics studies have determined the distribution ofquadruplex motifs in the human genome: i) The human genome has 370 000potential quadruplexes motifs, ii) 40% of the genes encoding a proteinhave at least one quadruplex located at 1 kb from initiation site of thetranscription, iii) important transcription factor binding sites, suchas SP1, MAZ, Krox ZF5 are positioned near or overlap the G4 motifs, iv)37% of the preferential recombination sites present a G4 motif. Thesecorrelations are conserved among different species in the evolution ofthe genomes.

Recently several in vivo studies have confirmed the existence of G4 inthe genomic DNA and cellular RNA. Some studies have used fluorescentprobes (antibodies or ligands) which specifically recognize G4s and donot bind to other DNA structures. These probes allowed the directdetection of G4s structures in immobilized chromosomes and confirmed thepresence of quadruplexes in the regulatory regions of genes andsubtelomeric regions. The involvement of G4s in replication,transcription, RNA splicing, or translation has also been extensivelystudied.

Quadruplexes have been identified as therapeutic targets, as follows:

a) Inhibition of Telomerase

Targeting G-quadruplexes was initiated in order to inhibit telomerase, aenzyme reactivated in 85% of cancers, but inactive in most normal cells.This enzyme recognizes the human telomeric sequence in itssingle-stranded conformation and maintains telomere length in tumorcells which makes them “immortal”. The strategy is to stabilizetelomeric G4s with chemicals to prevent telomerase interaction with itssubstrate. G4s ligands with anticancer properties have already beendiscovered, it is the case of Braco19, RHPS4 20 and 360A.

b) Inhibitions of Oncogenes

Many laboratories have been interested in quadruplex targeting toinhibit the expression of oncogenes. The oncogene c-myc is an importanttarget of this approach: it has a G4 sequence in its promoter and theexpression level of the gene depends on the formation of this structure.The formation of the G4 represses the transcription and this inhibitionis enhanced in the presence of G4 ligands. The same type of repressiveeffect of the ligand was observed for KRAS, c-kit and bcl2 oncogenes.

c) Targeting G4s

The unique features of the G4 topology, very distinct from a DNA or RNAduplex or single-strand, make it a therapeutic target. G4s are compactstructures which targeting can be likened to that of globular proteins.The great structural diversity of G4 suggests that a relatively highdegree of selectivity can be achieved. Examples of rational design ofligands and in silico screening are becoming more numerous in theliterature. This strategy opens a promising new era of targetingoffering an alternative to the usual proteins targeting strategy.Furthermore, if the first applications were related only to cancer, newapplications of this research are now considered in virology.

In a recent review, Harris et al proposed that the G4s may have abiological role in the life cycle of different pathogens (Harris &Merrick, PLoS Pathog. 2015 11(2):e1004562. The inventors also wrote areview describing the role of G4s in the replication cycle of manyviruses (Métifiot et al Nucleic Acid Res. 2014 42, 12352-66). In thecase of SARS coronavirus, a viral protein called “single domain SARS”(SUD) has two G4 binding sites. This viral protein seems essential forthe virulence of the virus, would fight the immune response of the hostby targeting the quadruplexes of the latter. In the case of Epstein-Barrvirus EBNA1 viral protein binds to the G4 DNA and is involved in theviral replication cycle. Finally, HPV also presents G4 sequences in itsgenome.

The RNA genome of HIV-1 is changing very quickly, which gives it animportant structural variability allowing it to escape the immuneresponse of the infected organism. The low fidelity of the reversetranscriptase and the many genetic recombination events between the twoviral RNAs are the drivers of this trend. These recombinations arefacilitated by the dimerization of the viral RNA at the DIS sequence(Dimer Initiation Site). A recent study has suggested that thisrecombination could also be done via a bimolecular quadruplex using thecPPT sequences of each of the viral RNAs. In another study, apreferential recombination site was found at the 5′ region of the genegag. This guanine-rich sequence is capable of forming a bimolecularquadruplex with the homologous sequence on the other strand. Thisquadruplex facilitates the exchange of material between the donor RNAand the recipient RNA. NCp7 protein is known to facilitate the packagingof the viral RNA, the reverse transcription and integration into thegenome, but is also able to open intramolecular RNA structures topromote the bimolecular structures. The NCp7 can also promote theformation of a bimolecular G4 with a receiver RNA.

Aptamers are nucleic acid sequences that can adopt a 3D structure andspecifically recognize a given target. These structured nucleic acidsare discovered by “SELEX” approaches. This is a method of in vitroselection from combinatorial libraries of synthetic oligonucleotidescontaining millions of different sequences. It is interesting to notethat during the last fifteen years many aptamers adopting G4 structureswere selected by this technique to target different HIV-1 proteins likethe integrase, RT, gp120 and Rev. Some of these aptamers can prevent theentry of the virus into the cell by interacting with the viral proteingp120. Concerning integrase the 93del aptamer potentially binds to thecatalytic pocket of the enzyme. This G4 strongly inhibits theinfectivity of HIV-1 with an IC₅₀ of 25 nM. A functional study of theeffects of this aptamer on the infectivity of HIV-1 in vivo conditionsdemonstrated that the fusion, transcription and integration of theprovirus are inhibited by 93del. These studies show that certain viralproteins recognize very specifically and with high affinity the G4structures.

In a previous application (EP14305763.6), the inventors showed that,despite its high genetic variability, HIV-1 genome presents several veryconserved G4 forming sequences. These G4 sequences are associated withcritical regulatory functions of the HIV replication cycle such as i)the initiation of reverse transcription by forming the centralinitiation point of the (+) strand synthesis by the reversetranscriptase; ii) the initiation of reverse transcription by formingthe first point of (+) strand synthesis by the reverse transcriptase;iii) the regulation of the transcription of the provirus. The HIV viruswas confronted to synthetic G4 DNA and RNA derived from its own genome.The observed inhibitory effects suggest that these synthetic “viral” G4sact as decoys diverting viral or cellular proteins from their naturaltargets in the viral genome.

US 2011/0064694 and US 2004/0116385 disclose porphyrin derivatives andtheir anti-viral activity. Porphyrin derivatives are disclosed interalliae by Sabater et al., Dalton transactions (2015), 44(8), 3701-3707and WO 200/27379. Perrone et al PLOSone 8, 8, e73121, 2013 discloses theantiviral effects of G-quadruplex binding in HIV.

According to a first object, the present invention concerns a compoundof formula (I):

Where

X represents a group selected from:

-   -   a mono or bi cyclic 5 to 10 membered heteroaryl or heterocycle        comprising at least one heteroatom chosen from N, O or S,        optionally substituted by a group chosen from OH, O(C1-C6)alkyl,        C1-C6 alkyl, Halogen, CN, NO₂, NRR′;    -   a guanidine group of formula

It being understood that X may be present in the form of an acid or baseaddition salt,

R and R′ identical or different independently represent a hydrogen atomor a group selected from C1-C6 alkyl, C6-C10 aryl, C3-C10 cycloalkyl,each alkyl, cycloalkyl and aryl being optionally substituted by one ormore of OH, O(C1-C6)alkyl, C1-C6 alkyl, C6-C10 aryl, Halogen, CN, NO₂,

. . . represents an optionally present single bond;

M is present or absent;

Where when . . . represents a single bond, M is present and represents ametal atom; together with a suitable counter ion, if appropriate,

for its use in the prevention and/or treatment of a viral infection.

According to an embodiment, X represents a guanidinium group of formula

According to an embodiment, X represents an optionally substitutedpyridine, such as a pyridine substituted by a C1-C6 alkyl, moreparticularly a methyl pyridinium.

Compounds H2-MA, Au-MA, Ni-PG, Au-PG, Mn-PG as depicted in FIG. 5 areparticularly suitable for the use of the invention.

According to an embodiment, the present invention also concerns the useof a compound of formula (I) according to the invention for thepreparation of a medicament for treating and/or preventing a viralinfection.

According to a further embodiment, the present invention also concerns amethod of treatment and/or prevention of a viral infection comprisingthe administration of a compound of formula (I) according to theinvention as defined above to a patient in the need thereof.

Viral infections include all disorders caused by virus which comprise Gquadruplex sequences in their genome at the DNA or RNA levels. Viralinfections include in particular HIV, Epstein Barr virus, HPV, SARScoronavirus, Ebola virus and Marburg virus. According to an embodiment,the compounds of formula (I) are those of formula (IA), such as those offormula (I′) or those of formula (I″).

According to a further object, the present invention concerns compoundsof formula (IA):

Where

A⁻ represents a counter anion;

M represents a metal atom, such as a gold atom (Au), cobalt atom (Co),Manganese (Mn) or a nickel (Ni) atom.

The present invention concerns in particular compounds H2-MA, Au-MA,Ni-PG, Au-PG, Mn-PG, more particularly Au-MA, Ni-PG, Au-PG, Mn-PG, asdepicted in FIG. 5.

According to a specific object, the present invention concerns compoundsof formula (I′):

Where

A⁻ represents a counter anion;

Au represents a gold atom,

and compounds of formula (I″):

Where

A⁻ represents a counter anion;

M represents a Co, Ni or Mn atom.

According to an embodiment, in formula (I), (IA), (I′) or (I″), A⁻represents an halogen ion, such as Cl⁻.

According to another object, the present invention concerns apharmaceutical composition comprising a compound of formula (IA) such as(I′) or (I″) according to the invention as defined above, together withat least one pharmaceutically acceptable excipient.

Unless specified otherwise, the terms used hereabove or hereafter havethe meaning ascribed to them below:

“Halo”, “hal” or “halogen” refers to fluorine, chlorine, bromine oriodine atom.

“Alkyl” represents an aliphatic-hydrocarbon group which may be straightor branched having 1 to 6 carbon atoms in the chain. In a particularlypreferred embodiment the alkyl group has 1 to 4 carbon atoms in thechain. Exemplary alkyl groups include methyl, ethyl, n-propyl,iso-propyl, iso-butyl, n-butyl, tert-butyl, n-pentyl, 3-pentyl.

“Cycloalkyl” refers to a non-aromatic mono- or polycyclic hydrocarbonring system of 3 to 10 carbon atoms. More preferably the cycloalkylgroup has of 4 to 10 carbon atoms, more preferably 4 to 8 carbon atomsand most preferably have 4 to 6 carbon atoms. Exemplary monocycliccycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and the like. Exemplary multicyclic cycloalkyl include1-decalin, norbornyl, adamant-(1- or 2-)yl.

“Aryl” refers to an aromatic monocyclic or bicyclic ring containing 6 to10 carbon atoms. Exemplary aryl groups include phenyl, naphthyl,indenyl, phenanthryl, biphenyl. Most preferably the aryl group isPhenyl.

The term “heteroaryl” refers to a 5 to 14, preferably 5 to 10 memberedaromatic mono-, bi- or multicyclic ring wherein at least one member ofthe ring is a hetero atom such as N, O, S. Examples include pyrrolyl,pyridyl, pyrazolyl, thienyl, pyrimidinyl, pyrazinyl, tetrazolyl,indolyl, quinolinyl, purinyl, imidazolyl, thienyl, thiazolyl,benzothiazolyl, furanyl, benzofuranyl, 1,2,4-thiadiazolyl, isothiazolyl,triazoyl, tetrazolyl, isoquinolyl, benzothienyl, isobenzofuryl,pyrazolyl, carbazolyl, benzimidazolyl, isoxazolyl.

The terms “heterocycle”, “heterocyclyl” or “heterocyclic” refer to asaturated or partially unsaturated non aromatic stable 3 to 14,preferably 5 to 10-membered mono, bi or multicyclic rings wherein atleast one member of the ring is a hetero atom, such as N, O, S.Typically, heteroatoms include, but are not limited to, oxygen,nitrogen, sulfur, selenium, and phosphorus atoms. Preferable heteroatomsare oxygen, nitrogen and sulfur. Suitable heterocycles are alsodisclosed in the Handbook of Chemistry and Physics, 76th Edition, CRCPress, Inc., 1995-1996, pages 2-25 to 2-26, the disclosure of which ishereby incorporated by reference. Preferred non aromatic heterocyclicinclude, but are not limited to oxetanyl, tetraydrofuranyl, dioxolanyl,tetrahydropyranyl, dioxanyl, pyrrolidinyl, piperidyl, morpholinyl,imidazolidinyl, pyranyl. Preferred saturated heterocycles are chosenfrom tetrahydrofuranyl, dioxolanyl, tetrahydropyranyl, dioxanyl,pyrrolidinyl, piperidyl, morpholinyl, imidazolidinyl, more preferablytetrahydrofuranyl, dioxolanyl, tetrahydropyranyl.

“metal” as used herein refers to Au, Ni, Mn, Co.

“Alkyl”, “cycloalkyl”, “aryl”, etc. . . . also refers to thecorresponding divalent “alkylene”, “cycloalkylene”, “arylene”, etc.,which are formed by the removal of two hydrogen atoms.

The compounds of formula (I) or (IA) such as (I′) or (I″) can beprovided in the form of a free base or in the form of addition saltswith acids, which also form part of the invention. The compounds of thepresent invention may possess an acidic group and a basic group whichmay form corresponding salts. Thus the present invention includes saltsof compounds of formula (I) or (IA) such as (I′) or (I″). The salts maypreferably be pharmaceutically acceptable salts. The acidic group mayform salts with bases. The base may be an organic amine base, forexample trimethylamine, tert-butylamine, tromethamine, meglumine,epolamine, etc. The acidic group may also form salts with inorganicbases like sodium hydroxide, potassium hydroxide, etc. The basic groupmay form salts with inorganic acids like hydrochloric acid, sulfuricacid, hydrobromic acid, sulfamic acid, phosphoric acid, nitric acid etcand organic acids like acetic acid, propionic acid, succinic acid,tartaric acid, citric acid, methanesulfonic acid, benzenesulfonic acid,glucoronic acid, glutamic acid, benzoic acid, salicylic acid,toluenesulfonic acid, oxalic acid, fumaric acid, maleic acid etc.Further, compounds of formula (I) or (IA) such as (I′) or (I″) may formquaternary ammonium salts and salts with amino acids such as arginine,lysine, etc. Lists of suitable salts may be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and P. H. Stahl, C. G. Wermuth, Handbook of Pharmaceuticalsalts—Properties, Selection and Use, Wiley-VCH, 2002, the disclosures ofwhich are hereby incorporated by reference.

These salts are advantageously prepared with pharmaceutically acceptableacids, but salts with other acids, useful for example for thepurification or for the isolation of the compounds of formula (I) or(IA) such as (I′) or (I″), also form part of the invention.

The compounds of formula (I) or (IA) such as (I′) or (I″) can compriseone or more asymmetric carbon atoms. They can therefore exist in theform of enantiomers or diastereoisomers. These enantiomers anddiastereoisomers, as well as their mixtures, including racemic mixtures,form part of the invention.

It is well known in the art how to prepare and isolate such opticallyactive forms. For example, mixtures of stereoisomers may be separated bystandard techniques including, but not limited to, resolution of racemicforms, normal, reverse-phase, and chiral chromatography, preferentialsalt formation, recrystallization, and the like, or by chiral synthesiseither from chiral starting materials or by deliberate synthesis oftarget chiral centers.

The compounds of formula (I) or (IA) such as (I′) or (I″) can also beprovided in the form of a hydrate or of a solvate, i.e. in the form ofassociations or combinations with one or more water or solventmolecules. Such hydrates and solvates also form part of the invention.

According to another object, the present invention concerns the processof preparation of a compound of formula (IA) such as (I′) or (I″)according to the invention as defined above.

The compounds and process of the present invention may be prepared in anumber of ways well known to those skilled in the art. The compounds canbe synthesized, for example, by application or adaptation of the methodsdescribed below, or variations thereon as appreciated by the skilledartisan. The appropriate modifications and substitutions will be readilyapparent and well known or readily obtainable from the scientificliterature to those skilled in the art. In particular, such methods canbe found in R. C. Larock, Comprehensive Organic Transformations, VCHpublishers, 1989 The reagents and starting materials may be commerciallyavailable, or readily synthesized by well-known techniques by one ofordinary skill in the arts. All substituents, unless otherwiseindicated, are as previously defined.

In the reactions described hereinafter, it may be necessary to protectreactive functional groups, for example hydroxy, amino, imino, thio orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Conventional protectinggroups herein named Pg may be used in accordance with standard practice,for examples see T. W. Greene and P. G. M. Wuts in Protective Groups inOrganic Synthesis, John Wiley and Sons, 1991; J. F. W. McOmie inProtective Groups in Organic Chemistry, Plenum Press, 1973.

Some reactions may be carried out in the presence of a base. There is noparticular restriction on the nature of the base to be used in thisreaction, and any base conventionally used in reactions of this type mayequally be used here, provided that it has no adverse effect on otherparts of the molecule. Examples of suitable bases include: sodiumhydroxide, potassium carbonate, triethylamine, alkali metal hydrides,such as sodium hydride and potassium hydride; alkyllithium compounds,such as methyllithium and butyllithium; and alkali metal alkoxides, suchas sodium methoxide and sodium ethoxide.

Usually, reactions are carried out in a suitable solvent. A variety ofsolvents may be used, provided that it has no adverse effect on thereaction or on the reagents involved. Examples of suitable solventsinclude: hydrocarbons, which may be aromatic, aliphatic orcycloaliphatic hydrocarbons, such as hexane, cyclohexane, benzene,toluene and xylene; amides, such as dimethyl-formamide; alcohols such asethanol and methanol and ethers, such as diethyl ether andtetrahydrofuran.

The reactions can take place over a wide range of temperatures. Ingeneral, it was found convenient to carry out the reaction at atemperature of from 0° C. to 150° C. (more preferably from about roomtemperature to 100° C.). The time required for the reaction may alsovary widely, depending on many factors, notably the reaction temperatureand the nature of the reagents. However, provided that the reaction iseffected under the preferred conditions outlined above, a period of from3 hours to 20 hours will usually suffice.

The compound thus prepared may be recovered from the reaction mixture byconventional means. For example, the compounds may be recovered bydistilling off the solvent from the reaction mixture or, if necessaryafter distilling off the solvent from the reaction mixture, pouring theresidue into water followed by extraction with a water-immiscibleorganic solvent and distilling off the solvent from the extract.Additionally, the product can, if desired, be further purified byvarious well-known techniques, such as recrystallization,reprecipitation or the various chromatography techniques, notably columnchromatography or preparative thin layer chromatography.

In particular, the compounds of the present invention may be preparedfrom the processes described below.

According to the an embodiment, the process of the invention maycomprise the step of reacting a compound of formula (II)

Where X is defined as in formula (I) (IA) such as (I′) or (I″) above,

Where B⁻ represents a counter anion, such as trifluoroacetate

With a gold-containing complex, such as a complex of formula (III)

KAu^(III)A₄  (III)

Where A⁻ is a counter anion as defined in formula (I′).

Generally, this step may be carried out in a suitable solvent such as aprotic and polar solvent, such as water or acetic acid.

Typically, this reaction may be conducted at a temperature comprisedbetween room temperature and the reflux temperature of the reactionmixture.

The process of the invention may further comprise the additional step ofisolating, purifying and/or formulating the compound of formula (IA)such as (I′) or (I″) following coupling of compounds (II) and (III).

Generally, the compounds of formula (III) are commercially available orcan be prepared by application or adaptation of known methods.

Compounds of formula (II) may be prepared according to Sabater et al.,Dalton transactions (2015), 44(8), 3701-3707.

More specifically, the compounds of formula (II) may be obtained byeither of the following pathways:

-   -   i) Where X represents a guanidine group: the compounds of        formula (II) may be prepared by reacting a compound of formula        (IV)

Where Pg represents a protecting group of the amino group, such a Bocwith the acid of formula (V):

H—B  (V)

Where B is defined as in formula (II).

Generally, this reaction may be carried out in an aprotic solvent, suchas dichloromethane.

The compounds of formula (IV) may be obtained from the compound offormula (VI):

With a compound of formula (VII):

Where Pg is defined as above.

This reaction may be conducted in a suitable solvent such as chloroform.

The porphyrin of formula (VI) is commercially available or may beprepared by application or adaptation of known procedures such as thosedisclosed by Bettelheim et al Inorg. Chem. 1987, 26, 1009-1017.

-   -   ii) Where X represents a methylpyridinium, the compound of        formula (II) may be prepared by reacting a compound of formula        (VIII)

With a compound of formula (IX):

Hal-CH₃  (IX)

Where Hal represents typically I.

Generally, this reaction is followed by the addition of the acid H—B(V).

The coupling reaction may be conducted in a suitable solvent such as DMFor acetone or their mixture and may typically be conducted at atemperature comprised between room temperature and the refluxtemperature of the reaction mixture.

The compound (VIII) may be obtained by reacting the compound of formula(X):

With a compound of formula (XI):

in a solvent such as propionic acid, at a temperature comprised betweenroom temperature and the reflux temperature of the reaction mixture.

As used herein, the term “patient” refers to a warm-blooded animal suchas a mammal, preferably a human or a human child, which is afflictedwith, or has the potential to be afflicted with one or more diseases andconditions described herein.

As used herein, a “therapeutically effective amount” refers to an amountof a compound of the present invention which is effective in reducing,eliminating, treating or controlling the symptoms of theherein-described diseases and conditions. The term “controlling” isintended to refer to all processes wherein there may be a slowing,interrupting, arresting, or stopping of the progression of the diseasesand conditions described herein, but does not necessarily indicate atotal elimination of all disease and condition symptoms, and is intendedto include prophylactic treatment and chronic use.

As used herein, the expression “pharmaceutically acceptable” refers tothose compounds, materials, compositions, or dosage forms which are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio.

The dosage of drug to be administered depends on such variables as thetype and extent of progression of the disease or disorder, the overallhealth status of the particular patient, the relative biologicalefficacy of the compound selected, and formulation of the compound,excipients, and its route of administration.

The compounds of present invention may be formulated into apharmaceutically acceptable preparation, on admixing with a carrier,excipient or a diluent, in particular for oral or parenteral use. Oralpreparations may be in the form of tablets, capsules or parenterals. Asolid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. Liquid carriers can includewater, an organic solvent, a mixture of both or pharmaceuticallyacceptable oils and fats. The compositions may conveniently beadministered in unit dosage form and may be prepared by any of themethods well known in the pharmaceutical art, for example, as describedin Remington: The Science and Practice of Pharmacy, 20th ed.; Gennaro,A. R., Ed.; Lippincott Williams & Wilkins: Philadelphia, Pa., 2000.Pharmaceutically compatible binding agents and/or adjuvant materials canbe included as part of the composition.

The tablets, pills, powders, capsules, troches and the like can containone or more of any of the following ingredients, or compounds of asimilar nature: a binder such as microcrystalline cellulose, or gumtragacanth; a diluent such as starch or lactose; a disintegrant such asstarch and cellulose derivatives; a lubricant such as magnesiumstearate; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, or methyl salicylate. Preferred tablets contain lactose,cornstarch, magnesium silicate, croscarmellose sodium, povidone,magnesium stearate, or talc in any combination. Capsules can be in theform of a hard capsule or soft capsule, which are generally made fromgelatin blends optionally blended with plasticizers, as well as a starchcapsule. In addition, dosage unit forms can contain various othermaterials that modify the physical form of the dosage unit, for example,coatings of sugar, shellac, or enteric agents. Other oral dosage formssyrup or elixir may contain sweetening agents, preservatives, dyes,colorings, and flavorings. In addition, the active compounds may beincorporated into fast dissolve, modified-release or sustained-releasepreparations and formulations, and wherein such sustained-releaseformulations are preferably bi-modal.

Liquid preparations for administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. The liquidcompositions may also include binders, buffers, preservatives, chelatingagents, sweetening, flavoring and coloring agents, and the like.Non-aqueous solvents include alcohols, propylene glycol, polyethyleneglycol, acrylate copolymers, vegetable oils such as olive oil, andorganic esters such as ethyl oleate. Aqueous carriers include mixturesof alcohols and water, hydrogels, buffered media, and saline. Inparticular, biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be useful excipients to control the release of the activecompounds. Intravenous vehicles can include fluid and nutrientreplenishers, electrolyte replenishers, such as those based on Ringer'sdextrose, and the like. Other potentially useful parenteral deliverysystems for these active compounds include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes.

Other features of the invention will become apparent in the course ofthe following description of exemplary embodiments that are given forillustration of the invention and not intended to be limiting thereof.

DESCRIPTION OF THE FIGURES

FIGS. 1 to 4 illustrate effect of the G4 ligands on HIV-1 replication.

FIG. 1: Effect of G4 ligands on G4 stability: The FRET meltingexperiments were performed in the presence of 0.5 μM of G4 ligand andusing 0.2 μM of HIV-PRO sequence labelled with fluorophores.

FIG. 2: Effects of G4 ligands compared with AZT on HIV-1 infectivityusing the HeLa P4 cell system for studying the replication of HIV-1. TheHeLa P4 cells contain a gene lacZ encoding beta-galactosidase under thecontrol of the viral LTR and whose transcription is activated by theviral protein Tat. The cells are incubated with the ligand then infectedwith HIV-1. After 24 hours the activity of beta-galactosidase, which isproportional to the infectivity of the virus is then measured on afluorescence plate reader.

FIG. 3: Correlation between the apparent affinity of the ligands for theG4 (expressed in stabilisation of the G4 in ΔTm ° C.) and theirinhibition activity on HIV.

FIG. 4: Measurement of the cell proliferation of HeLa cells as afunction of time in the presence of increasing the concentrations ofAu-MA G4 ligand.

FIG. 5 represents the structure of the compounds of the invention thatare tested and illustrated in FIG. 3. X═H2 refers to the case where nometal is in the center (M is absent).

FIG. 6 represents the structure of comparative prior art compounds thatare tested and illustrated in FIG. 3. X═H2 refers to the case where nometal is in the center (M is absent).

The above mentioned features of the invention are given for illustrationof the invention and not intended to be limiting thereof.

The following examples describe the synthesis of some compoundsaccording to the invention. These examples are not intended to belimitative and only illustrate the present invention.

EXAMPLES 1. Synthesis of Compounds of Formula (I) or (IA) 1.1.[Preparation of meso-tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl)porphyrinatogold(III) pentachloride] (Au-MA)

The synthesis of the non-metallated porphyrin was previously describedin Sabater et al. Dalton Trans. 2015, 44, 3701.

Step 1: Synthesis of 5,10,15,20-tetrakis(4-(pyrid-2-yl)phenyl)porphyrin(1)

4-(Pyrid-2-yl)benzaldehyde (2.8 g, 15.3 mmol) was dissolved in propionicacid (72 mL), pyrrole (1 g, 15.6 mmol) was added and the mixture wasrefluxed for 1 h in the dark. The solvent was evaporated and the residuedried under vacuum. The crude product was taken in dimethyl formamide(50 mL) and filtered. The product was washed with dimethyl formamide (50mL) and diethyl ether (2×50 mL) and dried under vacuum. Yield: 0.78 g(0.84 mmol, 22%) purple solid. ¹H NMR (250 MHz, CDCl₃) δ=8.99 (s, 8H,pyrrole), 8.90 (d, J=5 Hz, 4H, pyridine), 8.44 (d, J=8 Hz, 8H, phenyl),8.38 (d, J=8 Hz, 8H, phenyl), 8.10 (d, J=8 Hz, 4H, pyridine), 7.95 (ddd,J=8, 8, 1 Hz, 4H, pyridine), 7.40 (dd, J=8, 5 Hz, 4H, pyridine), −2.66(s, 2H, NH). TLC Rf≈0.20 (SiO₂, CH₃CN/H₂O/KNO₃ sat. 8/1/1).

Step 2: Synthesis of5,10,15,20-tetrakis(4-(N-methyl-pyridinium-2yl)phenyl)porphyrintetrakis(trifluoroacetate) (2)

5,10,15,20-Tetrakis(4-(pyrid-2-yl)phenyl)porphyrin (1) (200 mg, 0.22mmol) was dissolved in dimethylformamide (20 mL) and excess iodomethane(4 mL) was added. The mixture was heated at 155° C. for 3 h and acetone(100 mL) was added. The resulting purple precipitate was filtered off,washed with acetone, chloroform and diethyl ether. The product waspurified on reverse phase C18 column (20 g), elution water with 0.1% TFAthen water/acetonitrile, 80/20, v/v with 0.1% TFA. Yield: 210 mg (0.14mmol, 66%) purple solid. ¹H NMR (300 MHz, d⁶-DMSO) δ=9.33 (d, J=6 Hz,4H, pyridine), 9.04 (s, 8H, pyrrole), 8.84 (dd, J=8, 8 Hz, 4H,pyridine), 8.55 (d, J=8 Hz, 8H, phenyl), 8.48 (dd, J=8, 1 Hz, 4H,pyridine), 8.33 (ddd, J=8, 6, 1 Hz, 4H, pyridine), 8.19 (d, J=8 Hz, 8H,phenyl), 4.53 (s, 12H, CH₃−N), −2.83 (s, 2H, NH). UV-Vis (H₂O), λmax nm(ε M⁻¹ cm⁻¹) 416 (410×10³), 516 (15×10³), 552 (7×10³), 580 (5×10³), 634(3×10³). HRES⁺-MS m/z: calculated for [C₆₈H₅₄N₈]⁴⁺=245.6112, found:245.6106. TLC Rf≈0.15-0.20 (SiO₂, CH₃CN/H₂O/KNO₃ sat. 8/1/1).

Step 3:5,10,15,20-tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl)porphyrinatogold(III)pentachloride (Au-MA)

Tetrakis(4-(N-methyl-pyridinium-2-yl)phenyl)porphyrintetrakis(trifluoroacetate) (2) (30.5 mg, 0.021 mmol) was dissolved inwater (10 mL). NaOH 1 M, 0.1 mL was added. KAu^(III)Cl₄ (11.4 mg, 0.030mmol, 1.4 mol. eq.) was dissolved in water (1 mL) and added to theporphyrin solution. The mixture was refluxed for 24 h. The reaction wasmonitored by UV-visible spectroscopy and was stopped when the Soret bandshift was complete (from 438 to 406 nm, in water, acidic pH). Thereaction medium was cooled to room temperature. Desalting of theporphyrin was performed by reverse phase chromatography on a C18 Sep-Pakcartridge (5 g, Waters) by elution with water (200 mL) followed bymethanol (20 mL). The collected fractions were evaporated to dryness andproduct taken in methanol/water, 50:50, v/v (20 mL). Anion exchange wasperformed on a DOWEX 1×8-200 resin column (chloride form, 1 g). Theproduct solution was evaporated to dryness. The product was dissolved inmethanol and precipitated by the addition of diethyl ether. Aftercentrifugation the pellet was dried. Yield: 25.3 mg (0.0185 mmol, 88%)red solid. ¹H NMR (400 MHz, CD₃OD) δ=9.61 (s, 8H, pyrrole), 9.26 (d, J=6Hz, 4H, pyridine), 8.86 (dd, J=8, 8 Hz, 4H, pyridine), 8.66 (d, J=8 Hz,8H, phenyl), 8.51 (d, J=8 Hz, 4H, pyridine), 8.32-8.27 (m, 12H, pyridineand phenyl), 4.66 (s, 12H, CH₃—N). UV-Vis (H₂O), λmax nm (εM⁻¹ cm⁻¹) 406(400×10³), 520 (21×10³). ES⁺-MS m/z=235.49 [M-5 Cl]⁵⁺, 303.10 [M-4Cl]⁴⁺,415.80 [M-3Cl]³⁺. TLC Rf≈0.24 (SiO₂, CH₃CN/H₂O/KNO₃ sat. 6/1/1).

1.2. [Preparation of5,10,15,20-tetrakis(4-phenylguanidinium)porphyrinatogold(III)pentachloride] (Au-PG)

The synthesis of the non-metallated porphyrin was previously describedin Sabater et al. J. Biol. Inorg. Chem. 2015, 20, 729.

Step 1: Synthesis of5,10,15,20-tetrakis(4-(N,N′-ditertbutoxycarbonylphenylcarboxamidine)porphyrin(3)

Porphyrin 5,10,15,20-(tetra-4-aminophenyl)porphyrin (502 mg, 0.74 mmol)and N,N′-bis(tertbutyloxycarbonyl)pyrazole-1-carboxamidine (1.2 g, 3.8mmol) were dissolved in 15 mL of dry chloroform and the reaction mixturewas stirred at room temperature, under argon, for 5-7 days while beingmonitored by TLC (SiO₂, diethyl ether). After removal of the solventunder reduced pressure, the product was purified by silica gelchromatography. The column was eluted with 250 mL ofhexane/dichloromethane, 7/3, v/v with 1% triethylamine, followed by 500mL hexane/dichloromethane, 6/4, v/v with 1% triethylamine, and 500 mLhexane/dichloromethane, 50/50, v/v, 1% triethylamine. The fraction ofinterest was dried under reduced pressure. The solid residue was washedwith diethyl ether to ensure elimination of contaminating pyrazolederivative. Pure compound 3 was obtained as a purple solid (790 mg,65%): Rf=0.7 (SiO₂, hexane/ethyl acetate, 7/3); ¹H NMR (300 MHz, CDCl₃):δ=11.83 (s, 4H, N—H), 10.80 (s, 4H, N—H), 8.95 (s, 8H, pyrrole), 8.22(d, J=8 Hz, 8H, phenyl), 8.09 (d, J=8 Hz, 8H, phenyl), 1.65 (s, 36H,CH₃), 1.61 (s, 36H, CH₃), −2.75 (s, 2H, N—H porphyrin); ¹³C NMR (75 MHz,CDCl₃): δ=163.67, 153.69, 153.50, 138.50, 136.75, 135.06,131.23, 120.18,119.60, 83.93, 79.88, 28.26, 28.18 ppm; HRMS-ES+m/z [M+H]+ calcd forC₈₈H₀₇N₁₆O₁₆: 1643.8051 (95%), 1644.8081 (100%), found: 1643.8074 (95%),1644.8086 (100%). TLC Rf=0.7 (SiO₂, hexane/Et₂O 1/1).

Step 2: Synthesis of 5,10,15,20-tetrakis(4-phenylguanidinium)porphyrintetratrifluoroacetate (4)

Porphyrinmeso-5,10,15,20-tetrakis(4-(N,N′-ditertbutoxycarbonylphenylcarboxamidine)porphyrin(3) (800 mg, 0.49 mmol) was dissolved in 80 mL of dichlotomethane andreacted with 20 mL of trifluoroacetic acid under stirring for 3-4 h andthe reaction mixture was evaporated under reduced pressure. The productwas purified by dissolution in methanol followed by precipitation withdiethyl ether. Precipitation procedure was repeated 4 times. Theprecipitate was filtered on a fritted glass, washed with diethyl etherto provide 4 as a microcrystalline purple solid (592 mg, 93%); ¹H NMR(250 MHz, d⁶-DMSO): δ=10.33 (s, 4H, N—H), 9.01 (s, 8H, pyrrole), 8.28(d, J=8 Hz, 8H, phenyl), 7.87 (brs, 16H, N—H₂), 7.72 (d, J=8 Hz, 8H,phenyl), −2.90 (s, 2H, N—H porphyrin); HRMS-ES+m/z [M-4(CF₃CO₂)-3H]⁺calcd for C₄₈H₄₃N₁₆: 843.3857, found: 843.3873.

Step 3: 5,10,15,20-tetrakis(4-phenylguanidinium)porphyrinatogold(III)pentachioride (Au-PG)

Porphyrin meso-5,10,15,20-tetrakis(4-phenylguanidinium) porphyrintetratrifluoroacetate (4) (50.2 mg, 0.039 mmol) was dissolved in aceticacid 10 mL. KAuCl₄ (53.0 mg, 0.014 mmol) was dissolved in water 2 mL andadded to the porphyrin solution. The mixture was heated at 110° C. for24 h. The reaction was monitored by UV-visible spectroscopy and wasstopped when the Soret band shift was complete (Soret band of the themetallated derivative at 408 nm in acidic water). After evaporation todryness the product was taken in water/methanol, 80/20, v/v and themedium was centrifuged. The supernatant was loaded on a reverse phaseC18 Sep-Pak cartridge (5 g, Waters). Elution with water (200 mL) wasfollowed by methanol (20 mL) and then methanol containing 0.5%trifluoroacetic acid. The collected fractions were evaporated to drynessand product taken in methanol/water, 50/50, v/v (20 mL). Anion exchangewas performed on a DOWEX 1×8-200 resin column (chloride form, 1 g). Theproduct solution was evaporated to dryness. The product was dissolved inmethanol (5 mL) and precipitated by the addition of diethyl ether (20mL). After centrifugation the pellet was washed with diethyl ether anddried. Yield: 28.6 mg (0.023 mmol, 60%) red solid. ¹H NMR (400 MHz,CD₃OD): δ=9.54 (s, 8H, pyrrole), 8.41 (d, J=8 Hz, 8H, phenyl), 7.87 (d,J=8 Hz 8H, phenyl). UV/vis (H₂O): λmax nm (ε M⁻¹ cm⁻¹) 408 (285×10³).ES⁺-MS m/z=346.9 [M-2H-5Cl]³⁺, 260.2 [M−H-5Cl]⁴⁺.

2. Biological Activity 2.1. Methods

A. Preparation of the oligonucleotides:

Fluorescent oligonucleotides were purchased from Eurogentec (Seraing,Belgium) with “Reverse-Phase Cartridge Gold purification”.Concentrations were determined by ultraviolet (UV) absorption using theextinction coefficients provided by the manufacturer. Alloligonucleotides were dissolved in 20 mM potassium phosphate buffer pH7containing 70 mM KCl.

B. FRET melting experiments:

The HIV-PRO sequence (5′TGGCCTGGGCGGGACTGGG3′) (SEQ ID No 1) waslabelled with fluorescein at the 5′end and TAMRA at 3′end. The transferof fluorescence energy between fluorescein and tetramethylrhodamine isonly possible when the two fluorophores are close in the folded state atlow temperature. In the unfolded state at high temperature, the FRET isreduced. The fluorescence melting profiles were recorded on a Stratagenequantitative PCR device (De Cian A, et al. Fluorescence-based meltingassays for studying quadruplex ligands. Methods. 2007 June;42(2):183-95.).

C. Cell lines and viruses: HeLa P4 cells encoding a Tat-inducibleβ-galactosidase were maintained in DMEM medium (Invitrogen) supplementedwith 10% inactivated FCS, 1 mg/ml geneticin (G418, Gibco-BRL),gentamycin. MT4 and H9LaI cells were grown in RPMI 1640 glutamax medium(Invitrogen) supplemented with 10% inactivated FCS. HIV-1 viruses wereobtained after 48 h co-culture of MT4 cells (0.5×106/ml) and H9Laï cells(1×106/ml), chronically infected by HIV-1LaI isolate, in RPMI 1640glutamax medium supplemented with 10% inactivated FCS, at 37° C. underhumidified atmosphere and 5 CO2. The culture was then centrifuged andthe supernatant was clarified by filtration on a 0.45 μm membrane beforefreezing at −80° C.

D. Viral infectivity test: The G4 ligands are incubated in presence ofthe HelaP4 cells 20 minutes before infection. The infectivity wasassayed on HeLa P4 cells expressing CD4 receptor and the β-galactosidasegene under the control of the HIV-1 LTR. HeLa P4 were plated using 200μl of DMEM medium supplemented with 10% inactivated FCS in 96-multi-wellplates at 10 000 cells per well. After overnight incubation at 37° C.,under humidified atmosphere and 5% CO2, the supernatant was discardedand 200 μl of viral preparation were added in serial dilutions. After 24h of infection, the supernatant was discarded and the wells were washed3 times with 200 μl of PBS. Each well was refilled with 200 μl of areaction buffer containing 50 mM Tris-HCl pH 8.5, 100 mMβ-mercaptoethanol, 0.05% Triton X-100 and 5 mM4-methylumbelliferyl-B-D-galactopyranoside (4-MUG) (Sigma). After 24 h,the reaction was measured in a fluorescence microplate reader (CytofluorII, Applied Biosystems) at 360/460 nm Ex/Em.

E. Cytotoxicity study of the G4 ligands: HeLa, human epithelialcarcinoma cell line and Wi38, normal human fibroblast cell line wereused as experimental model to assess cellular proliferation in thepresence of G4 ligands. Cells have been seeded in 384-well plates. Allmeasures have been performed in duplicates in one experiment. The cellproliferation was measured as the surface of the well occupied by thecells. A proliferation index was then calculated by making a ratio onthe surface occupied at the time-point preceding the treatment. The cellcount is also assessed at the last time-point of the kinetics. CytolysisLyzed cells are stained with a nuclear marker, able to enter only cellswith compromised membranes. The number of lyzed cells per well isreported all along the kinetics. For the last time-point of thekinetics, a percentage of cytolysis is also computed after getting themaximum cytolysis for each well by permeabilizing cells.

2.2. Results: Specific G4 Ligands Inhibits HIV-1 Replication 2.2.1.G-Quadruplex Ligands

As part of the anti-cancer strategies described above, a wide variety ofquadruplex ligands have been developed. These molecules are very good G4specific structural probes. They bind very little to double-stranded andsingle-stranded DNA but recognize very well all types of G4 withdissociation constants in the order of tens to hundreds of nano-molar.The ability of some ligands (for instance XM14 and Br-360A) to bind toHIV-PRO3 sequence was evaluated using a stabilization test by FRET (FIG.1). 3 different families of G4 ligands were used: Salfens,Bisquinoliniums and porphyrins. This technique allows to selectmolecules which bind to HIVPRO labelled with fluorophores. In this test,thermal denaturation experiments were performed followed by fluorescenceand measure the fluorescence emission of fluorescein. Ligand binding tothe fluorescent oligonucleotide stabilizes the structure and theseresults in an increase in the temperature of half-dissociation of thelatter (FIG. 1). The more the T_(m) increases, the larger is theaffinity of the ligand for the target. Thus, in the example shown, theligand 360A further stabilizes the G4 as compared to XM14 compound; itsaffinity for the G4 is higher. These experiments were also performed inthe presence of a duplex competitor composed of 26 base pairs (data notshown). Thus, the binding selectivity of the ligand for the G4 incompetition with the duplex can be evaluated. If the stabilization ismaintained in the presence of 50 molar equivalents of non-fluorescentcompetitor duplex (600 equivalents when comparing the number of basepairs to the number of G4 tetrads), the ligand is considered to beselective, but if the stabilization disappears, the ligand is considerednot very selective, and this is the case for three porphyrins (H2PYMA,AUT and Tmpyp4). 43 ligands were tested, 23 were selected from the threedifferent chemical families that have interesting affinity andspecificity features to study their inhibitory effects on HIV.

2.2.2. Inhibition of HIV

The next step was therefore to test the effect of these ligands on theviral replication (FIG. 2) and see if there is a correlation between theability to stabilize the G4 in vitro (ΔT_(m)) and the effect on viralinfectivity in cellulo (FIG. 2). By testing these 23 ligands, it wasshown that there is a significant correlation between the affinity of aligand for the G4, whatever its chemical or structural nature(Bisquinolinium, porphyrins and Salen) and its inhibitory potential(FIG. 2). An increase of G4 stabilization by 7° C. directly translatesin an IC₅₀ which is divided by 10. Compounds in the LA, MALA, T, PYLAand PYMA series are porphyrins derivatives disclosed in the literatureand are tested as a comparative examples. The best compounds areporphyrin derivatives according to the invention of the Ma and PG serieshaving an IC₅₀ of around 100 nM. Under the same conditions, AZT has aninhibitory effect just below with an IC₅₀ of 40 nM. For H2PYMA, AUT andTmpyp4 G4 ligands, the competition FRET experiments demonstrated thatthey were much less specific to the G4 structures. These compounds alsobind to duplex and single-stranded DNA. They turned out to be weakinhibitors of viral replication. The low efficiency was interpreted bythe “dilution” on non-specific targets. Cytotoxicity test on KB cells,A549, MCF7, MRC5, HCT116 and HeLa P4 showed no cytotoxic effect of theseligands on a period of 92 h and a concentration of up to 30 μM (FIG. 4).This correlation between stabilization in vitro and in vivo inhibition(FIG. 3), associated to the high specificity of these ligands, and theabsence of cytotoxicity on human cells, suggest that the observedinhibition is due to the recognition of quadruplex structures of thevirus in the viral RNA or DNA.

What is claimed is:
 1. A method for the prevention and/or treatment of aviral infection comprising administering a compound of formula (I):

where X represents a group selected from: a mono or bicyclic 5 to 10membered heteroaryl or heterocycle comprising at least one heteroatomchosen from N, O or S, optionally substituted by a group chosen from OH,O(C1-C6)alkyl, C1-C6 alkyl, halogen, CN, NO₂, NRR′; a guanidine group offormula

it being understood that X may be present in the form of an acid or baseaddition salt, R and R′ identical or different independently represent ahydrogen atom or a group selected from C1-C6 alkyl, C6-C10 aryl, C3-C10cycloalkyl, each alkyl, cycloalkyl and aryl being optionally substitutedby one or more of OH, O(C1-C6)alkyl, C1-C6 alkyl, C6-C10 aryl, halogen,CN, NO₂, . . . represents an optionally present single bond; M ispresent or absent; where when . . . represents a single bond, M ispresent and represents a metal atom; together with a suitable counterion, if appropriate, to a patient in the need thereof.
 2. The methodaccording to claim 1, where the viral infection is chosen from the groupconsisting in HIV, Epstein Barr virus, HPV, SARS coronavirus, Ebolavirus and Marburg virus.
 3. The method according to claim 1, where theviral infection is HIV.
 4. The method according to claim 1, where thecompound of formula (I) is of formula (IA):

where

A⁻ represents a counter anion; M represents H₂, a gold atom (Au), cobalt(Co), Manganese (Mn) or a nickel (Ni) atom.
 5. The method according toclaim 4, where A⁻ represents a halogen ion.
 6. The method according toclaim 4, where A⁻ is Cl⁻.
 7. A compound of formula (IA):

where

A⁻ represents a counter anion; and M represents H₂, Manganese (Mn) or anickel (Ni) atom.
 8. The compound of formula (IA) according to claim 7,where A⁻ represents a halogen ion.
 9. The compound according to claim 7,where A⁻ is Cl⁻.
 10. A pharmaceutical composition comprising a compoundof formula (IA) according to claim 7, together with at least onepharmaceutically acceptable excipient.
 11. Process of preparation of acompound according to claim 7 comprising reacting a compound of formula(II)

where X is defined as in formula (IA) above, where B⁻ represents acounter anion, such as trifluoroacetate with a gold-containing complex,such as a complex of formula (III):KAu^(III)A₄  (III) where A is as defined in formula (I′)